Abstract Epigenetic transcriptional regulation is required for a myriad of developmental processes as well as being a significant contributor to human diseases. X-chromosome inactivation provides an experimentally tractable system for the dissection of epigenetic inheritance. X-inactivation results in the mitotically-heritable transcriptional inactivation of one X-chromosome in female mammals, thereby equalizing X-linked gene dosage between males and females. X-inactivation requires the Xist long non-coding RNA that is expressed only from the inactive X-chromosome. Current models posit that Xist RNA induction and coating of the X- chromosome in cis triggers a series of epigenetic events that culminates in X-inactivation. Notably, how Xist is selectively induced in females and not at all in males and how it triggers silencing are still unresolved questions. The objective of this proposal is to address how Xist expression and X-linked gene silencing are triggered during X-inactivation. Our central hypothesis, based on our published work and preliminary data, is that genes that escape X-inactivation function as dose-sensitive factors that induce Xist and, separately, potentiate X-linked gene silencing selectively in females. X-inactivation escapees are expressed from both X- chromosomes in XX females, including from the otherwise inactivated X-chromosome; hence, their expression is higher in females compared to XY males. We developed and employed a custom allele-specific RNA- sequencing pipeline to compile a list of the escape genes in mouse epiblast stem cells, which harbor an inactive-X. We then prioritized validated escapees that are predicted to be evolutionarily conserved and which function as transcriptional/chromatin regulators for a role in inducing X-inactivation. In this proposal, we systematically test the dose-dependent and biochemical activities of two of these escape genes in triggering Xist expression and X-linked gene silencing using unbiased and integrated high-throughput approaches. The results are expected to inform how genes along the length of the X-chromosome are silenced and why females undergo X-inactivation and males do not. The epigenetic factors and mechanisms that execute X-inactivation are known to overlap with those that regulate embryonic development and disease progression. Thus, understanding the cascade of epigenetic events that characterizes X-inactivation offers a window into identifying the common factors and mechanisms that establish epigenetic expression patterns broadly.